PROJECT SUMMARY/ABSTRACT Adipose expansion is necessary to accommodate chronic excess caloric intake and characterized by an increase in adipocyte size (hypertrophy) and number (hyperplasia; adipogenesis). Though obesity is related to AT lipid handling and storage capacity, the mechanisms underlying the link between obesity and the metabolic syndrome (MetS) are poorly understood. The AT expandability hypothesis postulates that the capacity for subcutaneous (subQ) adipose expansion is a significant determinant of metabolic health, as impaired adipogenesis (limited hyperplasia) may lead to ectopic lipid deposition in non-adipose organs, contributing to the development of obesity-associated diseases. Some in vitro studies report a higher population of small fat cells (i.e. hyperplasia) in individuals with MetS and type 2 diabetes. Data from two human overfeeding studies (one from our group) demonstrate that a smaller adipocyte size resulted in a greater impairment of insulin sensitivity with weight gain. We are the only group to assess in vivo adipogenesis in subQ AT via the incorporation of deuterium (2H) into adipose cells of obese women and show that higher adipocyte formation was associated with facets of impaired metabolic health. Our findings and others are contrary to the AT expandability hypothesis and provide evidence that higher (not lower) adipogenesis (i.e. hyperplasia) is associated with obesity-related disorders. Using a randomized controlled trial (RCT), we will examine the effects of a 9-week intervention on mechanisms of AT expandability. Overweight men and women will be randomized to 30% overfeeding (OF) or a weight stable Control (CTL) group. The objectives of the proposal are to test in vivo adipogenesis, using a validated 2H-labeling approach, and other mechanisms of subQ AT expansion in response to weight gain, and to assess the relationship of adipose expansion with changes in metabolic outcomes. The primary hypothesis is that higher adipogenesis in response to OF will be accompanied by increased visceral adiposity and ectopic lipid, reduced insulin sensitivity, and pathological AT remodeling in individuals with impaired subQ AT expansion. Therefore, despite hyperplasia in weight gainers, a limited storage capacity of adipocytes may facilitate impaired health outcomes. This is the first RCT to test the validity of the `AT expandability hypothesis'. Findings will provide new knowledge on the influence of adipose characteristics on the metabolic responses to dynamic changes in weight in humans.